Early stage development of pharmaceuticals, including drug discovery and screening, relies heavily on the use of cultured cell lines. Cell lines are used, for example, to test putative drug therapies in cells intended to mimic a normal or diseased state of an organism to be treated. With regard to respiratory diseases, one shortcoming of known cell lines is that the cultured cells may have characteristics that are very different from the actual cells (e.g., epithelial cells) that are found in vivo during an active disease state.
To address this issue, differentiated primary cell cultures have been used to mimic in vivo-like cellular characteristics. See, e.g., T. M. Krunkosky et al., Am. J. Respir. Cell. Mol. Biol. 22, 685-692 (2000) (normal human bronchial epithelial, “NHBE” cells). U.S. Pat. No. 5,364,785 to Mather et al. describes a clonal cell line with a single epithelial cell type that has characteristics of bronchial or bronchiolar epithelial cells. Unfortunately, these primary cells can be difficult to manipulate due to their low efficiency of transferability.
Other methods have established tracheo-bronchial epithelial cell lines that maintain their differentiated function in vitro by relying on viral transformation or immortalization by transfection with various oncogenes. For example, U.S. Pat. No. 4,885,238, issued to Reddel et al., discloses human bronchial epithelial cells capable of growth in culture after viral transformation. These cells were transformed with SV40 or adenovirus-12 SV40 hybrid virus, or with a recombinant plasmid containing portions of the Rous sarcoma virus. The use of viruses to alter cellular function can be disadvantageous, as viral infection per se affects epithelial cells, thus creating cells that are not the same as those normally present in the organism being studied.
Certain known differentiated mouse epithelial cell culture systems require a feeder layer of another cell type. For example, mouse tracheal epithelial cells have been grown in an air/liquid interface culture on top of a collagen gel on a semi-permeable membrane, with the entire membrane co-cultured over a layer of NIH3T3 fibroblasts. H. Chen et al., Respir. Crit. Care Med. 161, A150 (2000). These feeder cells are frequently altered in some way, and, thus, do not mimic other primary cell types that might be present in the airway in vivo. Other systems comprise mouse airway epithelial cells that are grown in a less-differentiated state. For example, mouse tracheal epithelial cells have been grown submerged on plastic in an undifferentiated state. C. B. Robinson and R. Wu, J. Tiss. Cult. Meth. 13, 95-102 (1991). However, such systems are not optimal for testing potential drug therapies that will be used in cells that are highly differentiated in vivo.
Davidson et al. have disclosed a primary culture model of differentiated murine tracheal epithelial cells. See, D. J. Davidson et al., Am. J. Physiol. Lung. Cell. Mol. Physiol. 279, L766-L778 (2000). However, the presence of functional mucus cells was not observed in the transmission electron microscopy studies described in the initial report of the system. Moreover, the culture system appears to be limited to only one embodiment in which the result is a single culture morphology favoring ciliary cell development. It would be more advantageous to have a system that may be achieved by more than one method and by which a number of cell morphologies could be produced, such as mucus-secreting cells. Finally, the Davidson et al. technique utilizes a serum substitute in place of serum. A more advantageous method would be free of both serum and serum substitutes, thus providing a more simple method by which to study, for example, signal transduction in the cultured cells.
A significant challenge faced by researchers in respiratory diseases is correlating results from animal cell culture experiments to human trials. One intermediate method presently available for moving a potential drug/therapy from cell culture to human trial is through use of xenograft systems. See, e.g., U.S. Pat. No. 5,667,766 to Wilson et al., which disclosure is incorporated by reference herein in its entirety. However, these xenograft systems do not accurately mimic either in vitro or in vivo situations. A need still remains for a system that will reliably and accurately translate results from animal cell culture studies to human applicability.
Another area of study for researchers in respiratory diseases are mucins. Mucins are a family of glycoproteins secreted by the epithelial cells including those at the respiratory, gastrointestinal and female reproductive tracts. Mucins are responsible for the viscoelastic properties of mucus and at least eight mucin genes are known. D. J. Thornton, et al., J. Biol. Chem. 272, 9561-9566 (1997). Mucociliary impairment caused by mucin hypersecretion and/or mucus cell hyperplasia leads to airway mucus plugging that promotes chronic infection, airflow obstruction and sometimes death. Many airway diseases such chronic bronchitis, chronic obstructive pulmonary disease, bronchiectacis, asthma, cystic fibrosis and bacterial infections are characterized by mucin overproduction. E. Prescott, et al., Eur. Respir. J., 8:1333-1338 (1995); K. C. Kim, et al., Eur. Respir. J., 10:1438 (1997); D. Steiger, et al. Am. J. Respir. Cell Mol. Biol., 12:307-314 (1995). Analysis of airway secretions has identified MUC5AC and MUC5B as the primary mucin constituents of the respiratory mucus gel. Generally, mucus hypersecretion/mucus cell hyperplasia is treated in two ways: physical methods to increase clearance and mucolytic agents. However, a need remains for agents and methods for reducing mucin production and treating the disorders associated with mucin hypersecretion. Therefore, systems and methods to screen compounds and treatments for mucus hypersecretion and related disorders are needed.